Team:XHD-Wuhan-A-China/Contribution
Contribution
In order to provide useful support and contribution to future iGEM, we have carried out the following two aspects of work:
1. Characterization of a related previous part BBa_J23100 supplemented with new data.
2. Provide new data from literature for the previous part BBa_J23100.
Characterization of a related previous part BBa_J23100
1. Design and purposes
Based on BBa_J23100, a genetic circuit BBa_J23100-B0034-amilCP-B0015 was constructed to characterize the function of this BBa_J23100 promoter. Since we use new microbial chassis organisms in our project, we need a suitable plasmid backbone to ensure that our gene circuit can be expressed correctly. After our search and BLAST, the combination of the J23100-amilCP gene circuit and the pBBR1MCS-2 plasmid backbone is a new combination, which did not appear in the previous iGEM project.
2. Methods
2.1 Construction of J23100- pBBR1MCS2
We construct the following gene circuit based on the principles of synthetic biology, as shown in Figure 1.
Figure 1. Constitution of J23100-amilCP gene circuit
We cloned the J23100 promoter from the J23100- pSB1C3 plasmid, and used homologous recombination to obtain the recombinant plasmid J23100- pBBR1MCS-2, and verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through PCR and enzyme digestion.
2.2 AmilCP expression under the control of J23100 with pBBR1MCS-2
The plasmid J23100-pBBR1MCS-2 containing the J23100 promoter was transformed into E. coli DH5α, and then cultured in LB liquid medium with shaking 200 rpm overnight. The next day, the bacterial solution was diluted 1:100 and suspended in LB liquid medium, and repeated three times. OD600 and OD588 every half hour was measured by microplate reader.
3. Results and Conclusion
We used the plasmid J23100- pBBR1MCS-2 as a template, and then used PCR to obtain the corresponding J23100- pBBR1MCS-2 fragment, as shown in Figure 2. The length of the PCR fragment is consistent with expectations.
Figure 2. The Gel electrophoresis of the J23100-amilCP fragment
Figure 3. The Gel electrophoresis of the colony PCR and restriction digestion verification
As shown in the figure 3 above, the arrow on the left refers to the result of plasmid digestion verification. You can see two clear bands after digestion, and there is a shallow band on top, which is the effect of incomplete digestion. The neat band on the right is the result of colony PCR verification. It is found that the band is clear, indicating that the plasmid has been successfully transformed.
Figure 4. The expression of amilCP changes over time
Figure 5. Sample characteristics during microplate reader testing
As shown in Figure 4 and Figure 5, the combination of J23100-amilCP and pBBR1MCS-2 can be expressed normally in E. coli, and it is found that the trend between OD600 and OD588 is basically the same, but the value of OD600 is slightly lower during the entire culture process. It can be seen from the figure that after introducing our plasmid, the bacteria began to enter the logarithmic growth phase after 2 hours, and entered the plateau phase at 5 hours, and the OD value was around 0.4. The OD value after entering the logarithmic phase and the plateau phase is much lower than that of Escherichia coli that does not carry the plasmid. This result shows that our plasmid will affect the growth of E. coli and put greater growth pressure on Escherichia coli, which is a new contribution to iGEM.
4. References
Xi, L. I. , Ling, D. , & Yan-Qiao, J. . (2005). Study of the action of cadmium on the growth of escherichia coli by microcalorimetry. Journal of Wuhan University of Technology.
Contribution to the previous part BBa_J23100 based on literature
Introduction
Cadaverine is 1,5-diaminopentane (DAP), which has been considered as the key monomer for a building block of polyamides (PAs) and competes with fossil-based resources. In this study, the constitutive promoter J23100 driven lysine decarboxylase (CadA) for cadaverine production was established and compared in different Escherichia coli strains.
Results from the original article
The original text of the result as follow:
3.2. Comparison of J23100 and T7 promoter on CadA expression
The strains JW and JWpI used J23100 promoter for CadA expression, while T7WpI and T7W used T7 promoter. Actually, WpI was a strain which has integrated a gene cluster of PLacI-T7RNAP on the chromosome at HK022 phage attack site. Because lacI repressor was neither in front of T7RNA polymerase cassette nor included in the plasmid of pSU-T7-CadA, the inducer IPTG was unnecessary. We can compare the constitutive orthogonality between T7RNAP and T7 promoter with the constitutive J23100 promoter in WpI. As a result, JW and JWpI attained a CadA activity of 75 g-DAP/g-DCW-h, but the activity of T7WpI was 27 g-DAP/g-DCW-h while no activity was detected in T7W (Fig. 2A). JW and JWpI have the similar CadA expression level, while a slightly lower expression in T7WpI and even no expression in T7W (i.e., absence of T7RNAP) (Fig. 2B), which was consistent to the result of protein expression. As no protein expression was observed in T7W, whole-cell biotransformation was conducted using the biocatalysts JW, JWpI and T7WpI. The initial DAP production by JW was 86 % at 0.5 h and reached 97 % at 2 h (Fig. 2C, JW). Although similar activity and expression were found in JW and JWpI, delayed DAP production was observed in JWpI, in which the initial production rate was only 57 % at 0.5 h and reached 95 % DAP production at 2 h (Fig. 2C, JW and JWpI). In the case of T7WpI, the DAP production was 48 % at 0.5 h and gradually reached 97 % at 4 h (Fig. 2C, T7WpI).
Table 1 lists the E. coli chassis used in this experiment. This paper re-characterizes the strength of the J23100 promoter in different E. coli chassis, which did not appear in the previous iGEM project.
References
Wwt, A. , Cyh, A. , Pyw, A. , Sfh, B. , Hyl, B. , & Sfl, B. , et al. (2021). Whole-cell biocatalyst for cadaverine production using stable, constitutive and high expression of l-lysine decarboxylase in recombinant escherichia coli w3110. Enzyme and Microbial Technology.
